Virus receptors are important host-cell determinants that govern viral tropism, spread, and disease pathogenesis. Understanding the molecular interactions between viruses and their cognate receptors provides significant insight into virus biology and disease. The human polyomavirus JC virus (JCV) is an important pathogen that infects the majority of the human population. While initial infection with JCV proves relatively asymptomatic, the virus establishes a persistent, life-long infection in the kidney. In immunocompromised individuals JCV can become reactivated and spread to the central nervous system (CMS). In the CMS, JCV infects oligodendrocytes, glial cells that provide support for neurons. JCV infection causes cytolytic destruction of oligodendrocytes leading to the fatal disease, Progressive Multifocal Leukoencephalopathy (PML). Approximately 5-8% of individuals with AIDS develop PML. The basic mechanisms by which JCV engages host-cell receptors and elicits signaling pathways to initiate an infectious cycle remains poorly understood. A combinatorial approach utilizing genetic, biochemical, and cell biological strategies will help to elucidate the mechanisms by which JCV-receptor interactions leads to infection and PML. Three integrated specific aims are proposed. The first aim will determine the molecular mechanisms by which JCV engages the serotonin receptor 5-HT2AR to mediate viral attachment and entry. The second aim will define the signaling pathways activated in response to JCV infection, and explore the role of 5-HT2AR in JCV-induced signaling pathways. The third aim will investigate the role of PDZ-domain proteins in JCV infection. Public Health Relevance: Taken together, these studies will enhance our understanding of JCV-host-cell tropism and the molecular mechanisms underlying the pathogenesis of PML.